Miniaturized Synthetic Palmitoylation Motifs for Small-Molecule Localization in Living Cells.

Conjugating small-molecule ligands to synthetic motifs that can localize to specific organelles or membranes in living cells is a practical approach to develop compounds as chimeric tools or drugs that can manipulate biological processes in a subcellular site-specific manner. However, the number of available organelle-targeted synthetic motifs for small-molecule localization is limited. We have recently developed a synthetic myristoyl-DCys motif for small-molecule localization that undergoes S-palmitoylation via the cellular palmitoylation machinery and localizes to the Golgi surface. Herein, we show that the lipid acyl chain of the myristoyl (C14)-DCys motif can be as short as 10-carbons and still retain the palmitoylation-dependent Golgi localization property in cells. This discovery led to the identification of four new derivatives for small-molecule localization: tridecanoyl (C13)-, dodecanoyl (C12)-, undecanoyl (C11)-, and decanoyl (C10)-DCys motifs. We demonstrated that even the short decanoyl-DCys palmitoylation motif could be used to generate small-molecule ligand conjugates that functioned as chemical tools for controlling protein localization and cell signaling. The miniaturized synthetic palmitoylation motifs identified in this work may find applications in creating various Golgi-localizable chimeric molecules for use in chemical biology and drug development.

Revisit of the plasmon-mediated chemical transformation of para-aminothiophenol.

Fingerprint Raman features of para-aminothiophenol (pATP) in surface-enhanced Raman scattering (SERS) spectra have been widely used to measure plasmon-driven catalytic activities because the appearance of characteristic spectral features is purported to be due to plasmon-induced chemical transformation of pATP to trans-p,p'-dimercaptoazobenzene (trans-DMAB). Here, we present a thorough comparison of SERS spectra for pATP and trans-DMAB in the extended range of frequencies covering group vibrations, skeletal vibrations, and external vibrations under various conditions. Although the fingerprint vibration modes of pATP could be almost mistaken with those of trans-DMAB, the low-frequency vibrations revealed distinct differences between pATP and DMAB. Photo-induced spectral changes of pATP in the fingerprint region were explained well by photo-thermal variation of the Au-S bond configuration, which affects the degree of the metal-to-molecule charge transfer resonance. This finding indicates that a large number of reports in the field of plasmon-mediated photochemistry must be reconsidered.

A Peptide Nanocage Constructed by Self-Assembly of Oligoproline Conjugates.

Cage-like supramolecular assemblies called molecular cages, which possess attractive functions, have been prepared. Although biomolecule-based nanocages are required for biological/medical applications such as drug delivery systems, the majority of nanocages are constructed using aromatic compounds with lower biocompatibility and biodegradability. In this study, the construction of a peptide nanocage consisting of an oligoproline conjugate is demonstrated. The conjugate was easy to prepare and had high biocompatibility. The oligoproline moiety of the conjugate had a rigid, rod-like structure suitable for the backbone of the supramolecular nanocage. The conjugates self-assembled to form peptide nanocages with a huge inner cavity.

A photoactivatable self-localizing ligand with improved photosensitivity for chemo-optogenetic control of protein localization in living cells.

Light-mediated control of protein localization in living cells is a powerful approach for manipulating and probing complex biological systems. By incorporating a classical 6-nitroveratryloxycarbonyl (NVOC) caging group into the inner plasma membrane (PM)-localizing trimethoprim ligand, we recently developed a photoactivatable self-localizing ligand (paSL) that can rapidly recruit engineered Escherichia coli dihydrofolate reductase-fusion proteins from the cytoplasm to the PM upon violet (ca. 400 nm)-light illumination. However, because the photosensitivity of the NVOC-caged paSL is low to moderate, photouncaging experiments require high light intensity, which may not be ideal for many cell applications. Herein, we present a new 7-diethylaminocoumarin (DEAC)-caged paSL with improved photosensitivity. DEAC-caged paSL induced efficient protein recruitment upon violet-light irradiation, even at the low intensity under which NVOC-caged paSL does not respond. DEAC-caged paSL was insensitive to excitation light used to image green fluorescent proteins (GFPs), and it was applicable for simultaneous optical stimulation of Gαq signaling and fluorescence imaging of subsequent Ca2+ oscillations using a GFP-based Ca2+ biosensor in living cells.

A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution.

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells.

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

Chemogenetic Control of Protein Anchoring to Endomembranes in Living Cells with Lipid-Tethered Small Molecules.

The self-localizing ligand-induced protein translocation (SLIPT) system is an emerging platform that controls protein localization in living cells using synthetic self-localizing ligands (SLs). Here, we report a chemogenetic SLIPT system for inducing protein translocation from the cytoplasm to the surface of the endoplasmic reticulum (ER) and Golgi membranes, referred to as endomembranes. By screening a series of lipid-trimethoprim (TMP) conjugates, we found oleic acid-tethered TMP (oleTMP) to be the optimal SL that efficiently relocated and anchored Escherichia coli dihydrofolate reductase (eDHFR)-fusion proteins to endomembranes. We showed that oleTMP mediated protein anchoring to endomembranes within minutes and could be reversed by the addition of free TMP. We also applied the endomembrane SLIPT system to artificially activate endomembrane Ras and inhibit the active nuclear transport of extracellular signal-regulated kinase (ERK), demonstrating its applicability for manipulating biological processes in living cells. We envision that the present oleTMP-based SLIPT system, which affords rapid and reversible control of protein anchoring to endomembranes, will offer a new unique tool for the study and control of spatiotemporally regulated cell signaling processes.

Development of Cell-Penetration PG-Surfactants and Its Application in External Peptide Delivery to Cytosol.

Recently, development of techniques to deliver pharmacologically active biomacromolecules such as peptides and proteins to cytosol has gained much interest. Here, we applied the peptide gemini (PG)-surfactants to a novel platform to design cell penetration lipopeptides (CP-PGs), which can deliver exogenous peptides and proteins to cytosol. Among the number of candidate CP-PGs having different peptide sequences at the X-, Y-, and Z-positions, we focused on those having two C12 alkyl chains appended to the side chain of two Cys residues, the betaine sequence -Asp-Lys-Asp-Lys- between the alkylated Cys residues (i.e., at the X-position), and having different cationic peptide sequences of oligo-Lys or oligo-Arg at the Y- and/or Z-positions. With respect to cytotoxicity for mammalian cells such as NIH3T3 cells upon 1 h exposure, those having (Lys)3 (K3-DKDKC12 and DKCK12-K3) showed lower cytotoxicity (IC50 = 241 and 198 µM) among those having oligo-Lys, (Lys)n (n = 1, 3, 5; IC50 = 88-197 µM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys)3 at both N- and C-terminal sides (K3-DKDKC12-K3) (IC50 = 225 µM). In contrast, the CP-PG having (Arg)3 at the N-terminal side (R3-DKDKC12) showed higher cytotoxicity (IC50 = 88 µM). Carrier abilities of the CP-PGs for exogenous peptides were evaluated using the proapoptotic domain (PAD) peptide, which induces apoptosis by disturbing mitochondrial membranes after delivery into cytosol. As a result, the CP-PGs of K3-DKDKC12, DKCK12-K3, K3-DKDKC12-K3, DKCK12-K5, and R3-DKDKC12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC50) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 µM, respectively). Especially, the carrier abilities of DKCK12-K3 and DKCK12-K5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC50 = 6.8 µM). Together, our results indicate that the PG-surfactant molecular framework could be a potential new platform to design efficient cell penetration carrier materials.

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein.

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405nm, the hoeFL-DNA complex displayed two fluorescence bands around 460nm and 520nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F520/F460) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.

A Set of Organelle-Localizable Reactive Molecules for Mitochondrial Chemical Proteomics in Living Cells and Brain Tissues.

Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases.

ORP9-PH domain-based fluorescent reporters for visualizing phosphatidylinositol 4-phosphate dynamics in living cells.

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER-PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology.

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells.

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo , we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

Fluorophore labeling of native FKBP12 by ligand-directed tosyl chemistry allows detection of its molecular interactions in vitro and in living cells.

Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.

Synthetic self-localizing ligands that control the spatial location of proteins in living cells.

Small-molecule ligands that control the spatial location of proteins in living cells would be valuable tools for regulating biological systems. However, the creation of such molecules remains almost unexplored because of the lack of a design methodology. Here we introduce a conceptually new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target proteins and relocate (tether) them rapidly from the cytoplasm to their targeting sites, thus serving as synthetic protein translocators. SLL-induced protein translocation enables us to manipulate diverse synthetic/endogenous signaling pathways. The method is also applicable to reversible protein translocation and allows control of multiple proteins at different times and locations in the same cell. These results demonstrate the usefulness of SLLs in the spatial (and temporal) control of intracellular protein distribution and biological processes, opening a new direction in the design of small-molecule tools or drugs for cell regulation.

Chemo- and opto-genetic tools for dissecting the role of membrane contact sites in living cells: Recent advances and limitations.

Membrane contact sites (MCSs) are morphologically defined intracellular structures where cellular membranes are closely apposed. Recent progress has significantly advanced our understanding of MCSs with the use of new tools and techniques. Visualization of MCSs in living cells by split fluorescence proteins or FRET-based techniques tells us the dynamic property of MCSs. Manipulation of MCSs by chemically-induced dimerization (CID) or light-induced dimerization (LID) greatly contributes to our understanding of their functional aspects including inter-organelle lipid transport mediated by lipid transfer proteins (LTPs). Here we highlight recent advances in these tools and techniques as applied to MCSs, and we discuss their advantages and limitations.

Chemogenetic Manipulation of Endogenous Proteins in Fission Yeast Using a Self-Localizing Ligand-Induced Protein Translocation System.

Cells sense extracellular stimuli through membrane receptors and process information through an intracellular signaling network. Protein translocation triggers intracellular signaling, and techniques such as chemically induced dimerization (CID) have been used to manipulate signaling pathways by altering the subcellular localization of signaling molecules. However, in the fission yeast Schizosaccharomyces pombe, the commonly used FKBP-FRB system has technical limitations, and therefore, perturbation tools with low cytotoxicity and high temporal resolution are needed. We here applied our recently developed self-localizing ligand-induced protein translocation (SLIPT) system to S. pombe and successfully perturbed several cell cycle-related proteins. The SLIPT system utilizes self-localizing ligands to recruit binding partners to specific subcellular compartments such as the plasma membrane or nucleus. We optimized the self-localizing ligands to maintain the long-term recruitment of target molecules to the plasma membrane. By knocking in genes encoding the binding partners for self-localizing ligands, we observed changes in the localization of several endogenous molecules and found perturbations in the cell cycle and associated phenotypes. This study demonstrates the effectiveness of the SLIPT system as a chemogenetic tool for rapid perturbation of endogenous molecules in S. pombe, providing a valuable approach for studying intracellular signaling and cell cycle regulation with an improved temporal resolution.

Palmitoylation-Dependent Small-Molecule Fluorescent Probes for Live-Cell Golgi Imaging.

Small-molecule fluorescent probes enabling visualization of the Golgi apparatus in living cells are essential tools for studying Golgi-associated biological processes and diseases. So far, several fluorescent Golgi stains have been developed by linking ceramide lipids to fluorophores. However, ceramide-based probes suffer from cumbersome staining procedures and low Golgi specificity. Here, we introduce fluorescent Golgi-staining probes based on the tri-N-methylated myristoyl-Gly-Cys (myrGC3Me) motif. The cell-permeable myrGC3Me motif localizes to the Golgi membrane upon S-palmitoylation. By modularly conjugating the myrGC3Me motif to fluorophores, we developed blue, green, and red fluorescent Golgi probes, all of which allowed simple and rapid staining of the Golgi in living cells with high specificity and no cytotoxicity. The probe was also applicable to the visualization of dynamic changes of the Golgi morphology induced by drug treatments and during cell division. The present work provides an entirely new series of live-cell Golgi probes useful for cell biological and diagnostic applications.

Native FKBP12 engineering by ligand-directed tosyl chemistry: labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells.

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.